WO2023035027A1

WO2023035027A1 - Biocement and method of biocementation

Info

Publication number: WO2023035027A1
Application number: PCT/AU2022/051079
Authority: WO
Inventors: Abhijit Mukherjee; Navdeep DHAMI
Original assignee: Curtin University
Priority date: 2021-09-07
Filing date: 2022-09-06
Publication date: 2023-03-16
Also published as: AU2022343407A1

Abstract

The invention relates to a method of biocementation comprising applying at a location of a granular substrate a first component comprising an alkalophilic ureolytic calcifying bacterium and a soluble Ca2+ salt in a medium of pH 10 to about pH 13 and applying to the location of the granular substrate a second component comprising urea, a soluble Ca2+ salt, and yeast extract, wherein the first component is applied to the location of the granular substrate separately to the second component.

Description

Biocement and method of biocementation

Field

The invention relates to a method of biocementation, a kit for performing the method, bacteria for performing the method, a kit comprising the bacteria, and a biocement formed using the method or bacteria.

Background

In the last decade, bio-mimicked technology of bacterially induced calcium carbonate precipitation has been discovered for construction and repair of the infrastructure in a sustainable way. A major factor that limits the application of bacterial cement (biocement) in granular materials is non-uniform cementation, leading to poor performance. The attachment of cells throughout the substrate in a uniform way plays a crucial role in determining the performance of cemented structures and, therefore, needs to be optimised. Construction or repair and the dosage of biocement reagents need to be optimised, depending upon the application.

A range of bacterial pathways and cultures have been explored for biocement applications, however amongst all, the ureolytic bacterial strain Sporosarcina pasteurii ATCC 11851 has been found to be the most applicable culture. Extremely harsh environments, such as pH 13 in concrete and high salt contents in marine conditions, impedes the efficacy of the process of biocementation.

There is a need for improved methods and reagents for biocementation.

Summary

A first aspect provides a method of biocementation, comprising: a. applying to a location of a granular substrate a first component comprising an alkalophilic ureolytic calcifying bacterium and a soluble Ca²⁺ salt in a medium having about pH 10 to about pH 13; b. applying to the location of the granular substrate a second component comprising urea, a soluble Ca²⁺ salt, and yeast extract, wherein the first component is applied to the location of the granular substrate separately to the second component. A second aspect provides a kit when used according to the method of the first aspect, the kit comprising a first component comprising an alkalophilic ureolytic calcifying bacterium and CaCb and a second component comprising urea, a soluble Ca²⁺ salt, optionally CaCb, and yeast extract.

A third aspect provides a kit comprising a first component comprising an alkalophilic ureolytic calcifying bacterium and a soluble Ca²⁺ salt, optionally CaCb, and a second component comprising urea, a soluble Ca²⁺ salt, optionally CaCb, and yeast extract, wherein the alkalophilic ureolytic calcifying bacterium is Bacillus clausd' S111 deposited as accession number V20/026707 or Bacillus clausd' T09054 deposited as accession number V20/026706.

A fourth aspect provides a biocement when formed according to the method of the first aspect.

A fifth aspect provides a biocement comprising alkalophilic ureolytic calcifying bacterium Bacillus clausd' S111 deposited as accession number V20/026707 or Bacillus clausd' T09054 deposited as accession number V20/026706.

A sixth aspect provides Bacillus clausd' T09054 deposited as accession number V20/026706.

A seventh aspect provides Bacillus clausd' S111 deposited as accession number V20/026707.

Brief description of the drawings

Figure 1 is a dot plot illustrating the growth of Bacillus clausd' S111 ( ), Bacillus clausd'

T0905 (■ ), Sporosarcina pasteurd' grown in AIM ( A ), and Sporosarcina pasteurd' grown in HIM (O ) recorded over 48 hours at intervals of every 4 hours at an optical density of 600 nm.

Figure 2 is a bar graph illustrating the viability of Bacillus clausd' S111, Bacillus clausd' T0905, Sporosarcina pasteurd' grown in AIM, and Sporosarcina pasteurd' grown in HIM after 5 days using BacTiter-Glo™ (Promega) using luminescence.

Figure 3 is a bar graph illustrating the urease activity of Bacillus clausd' S111, Bacillus clausd' T0905, Sporosarcina pasteurd' grown in AIM, and Sporosarcina pasteurd' grown in HIM recorded over 5 days at intervals of every 24 hours.

Figure 4 is a dot plot illustrating the level of soluble calcium ions in the supernatant of cultures of Bacillus clausd' S111 ( ), Bacillus clausd' T0905(> ), Sporosarcina pasteurd' grown in AIM ( A ), and Sporosarcina pasteurii grown in HIM (O ) recorded over 24 hours at intervals of every 4 hours.

Figure 5 is a bar graph illustrating the concentration of insoluble CaCOs produced by Bacillus claussi S111, Bacillus claussi T0905, Sporosarcina pasteurii grown in AIM, and Sporosarcina pasteurii grown in HIM after 5 days growth.

Detailed description

The present invention relates to a method of application of specially formulated cement containing bacterial cells in all types of granular substrates such as concrete, sand, and soil, ensuring uniform biocementation in order to achieve high compressive strength, high resistance to permeation of deleterious materials, and enhanced durability. Improved application and attachment can lead to significant improvement in biocementation throughout the granular substrate resulting in improved quality control, high compressive strength and durability.

The inventors have developed a method of application of urease producing a Ikalophilic and halo-a Ikalophilic bacterial cultures for biocementation. These biocementing cultures offer high viability under extremophilic environments with high pH (pH 10-13) or and salt percentage (pH 10, NaCI 2-10 %). Disclosed herein are isolated urease producing alkalophilic and halophilic bacterial cultures as pure strains and in consortium, to be used as an additive to cement, or in isolation, for surface protection, crack healing and strength enhancement of concrete. The inventors have isolated two alkalophilic bacteria: Bacillus clausd' S111; and Bacillus clausd' T0905. These cultures can be used individually, in consortium as well as synergistically with S. pasteurii with significantly improved biocementation in different granular substrate materials. Also disclosed are protocols for developing bespoke solutions for soil and cementitious applications using biocement. Also disclosed is a carrier for supplementation of bacterial cells and spores.

A first aspect provides a method of biocementation, comprising: a. applying to a location of a granular substrate a first component comprising an alkalophilic ureolytic calcifying bacterium and a soluble Ca²⁺ salt in a medium having about pH 10 to about pH 13; b. applying to the location of the granular substrate a second component comprising urea, a soluble Ca²⁺ salt, and yeast extract or nutrient broth, wherein the first component is applied to the location of the granular substrate separately to the second component.

The first and second components work in combination. However, the first and second components are applied temporally separated. In one embodiment, the second component may be applied about 1 h, about 2 h, about 3 h, about 4 h, about 5 h, about 6 h, about 7 h, about 8 h, about 9 h, about 10 h, about 11 h, about 12 h, about 13 h, about 14 h, about 15 h, about 16 h, about 17 h, about 18 h, about 19 h, about 20 h, about 21 h, about 22 h, about 23 h, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 2 years, about 3 years, or more than 3 years.

In one embodiment, a first component is sprayed with a second component and mixed with a binder at a ratio from about 0.1 to about 1 % w/w. In one embodiment, the mixture is at a ratio from about 0.2 to about 1 % w/w, about 0.3 to about 1 % w/w, about 0.4 to about 1 % w/w, about 0.5 to about 1 % w/w, about 0.6 to about 1 % w/w, about 0.7 to about 1 % w/w, about 0.8 to about 1 % w/w, or about 0.9 to about 1 % w/w. In one embodiment, the bacterium is a Ika lophilic ureolytic calcifying bacterium Bacillus clausd' S111 deposited as accession number V20/026707 or Bacillus clausd' T09054 deposited as accession number V20/026706.

In one embodiment, a soluble Ca²⁺ salt may comprise CaCb, Ca NOsh, CaSC , Ca(C2HsO2)2, or a combination thereof.

In one embodiment, the pH may be about pH 8.0, about pH 8.1, about pH 8.2, about pH 8.3, about pH 8.4, about pH 8.5, about pH 8.6, about pH 8.7, about pH 8.8, about pH 8.9, about pH 9.0, about pH 9.1, about pH 9.2, about pH 9.3, about pH 9.4, about pH 9.5, about pH 9.6, about pH 9.7, about pH 9.8, about pH 9.9, about pH 10.0, about pH 10.1, about pH 10.2, about pH 10.3, about pH 10.4, about pH 10.5, about pH 10.6, about pH 10.7, about pH 10.8, about pH 10.9, about pH 11.0, about pH 11.1, about pH 11.2, about pH 11.3, about pH 11.4, about pH 11.5, about pH 11.6, about pH 11.7, about pH 11.8, about pH 11.9, about pH 12.0, about pH 12.1, about pH 12.2, about pH 12.3, about pH 12.4, about pH 12.5, about pH 12.6, about pH 12.7, about pH 12.8, about pH 12.9, about pH 13.0, about pH 13.1, about pH 13.2, about pH 13.3, about pH 13.4, or about pH 13.5. In one embodiment, the NaCI may be about 0.5 % w/w, about 0.6 % w/w, about 0.7 % w/w, about 0.8 % w/w, about 0.9 % w/w, 1.0 % w/w, about 1.1 % w/w, about 1.2 % w/w, about 1.3 % w/w, about 1.4 % w/w, 1.5 % w/w, about 1.6 % w/w, about 1.7 % w/w, about 1.8 % w/w, about 1.9 % w/w, about 2.0 % w/w, about 2.1 % w/w, about 2.2 % w/w, about 2.3 % w/w, about 2.4 % w/w, about 2.5 % w/w, about 2.6 % w/w, about 2.7 % w/w, about 2.8 % w/w, about 2.9 % w/w, about 3.0 % w/w, about 3.1 % w/w, about 3.2 % w/w, about 3.3 % w/w, about 3.4 % w/w, about 3.5 % w/w, about 3.6 % w/w, about 3.7 % w/w, about 3.8 % w/w, about 3.9 % w/w, about 4.0 % w/w, about 4.1 % w/w, about 4.2 % w/w, about 4.3 % w/w, about 4.4 % w/w, about 4.5 % w/w, about 4.6 % w/w, about 4.7 % w/w, about 4.8 % w/w, about 4.9 % w/w, about 5.0 % w/w, about 5.1 % w/w, about 5.2 % w/w, about 5.3 % w/w, about 5.4 % w/w, about 5.5 % w/w, about 5.6 % w/w, about 5.7 % w/w, about 5.8 % w/w, about 5.9 % w/w, about 6.0 % w/w, about 6.1 % w/w, about 6.2 % w/w, about 6.3 % w/w, about 6.4 % w/w, about 6.5 % w/w, about 6.6 % w/w, about 6.7 % w/w, about 6.8 % w/w, about 6.9 % w/w, about 7.0 % w/w, about 7.1 % w/w, about 7.2 % w/w, about 7.3 % w/w, about 7.4 % w/w, about 7.5 % w/w, about 7.6 % w/w, about 7.7 % w/w, about 7.8 % w/w, about 7.9 % w/w, about 8.0 % w/w, about 8.1 % w/w, about 8.2 % w/w, about 8.3 % w/w, about 8.4 % w/w, about 8.5 % w/w, about 8.6 % w/w, about 8.7 % w/w, about 8.8 % w/w, about 8.9 % w/w, about 9.0 % w/w, about 9.1 % w/w, about 9.2 % w/w, about 9.3 % w/w, about 9.4 % w/w, about 9.5 % w/w, about 9.6 % w/w, about 9.7 % w/w, about 9.8 % w/w, about 9.9 % w/w, about 10.0 % w/w, about 10.1 % w/w, about 10.2 % w/w, about 10.3 % w/w, about 10.4 % w/w, or about 10.5 % w/w.

In one embodiment, the pH and NaCI percentage may be any combination of values from the values above.

In one embodiment, the bacterium is lyophilised.

In one embodiment, the first component comprises the bacterium and soluble Ca²⁺ salt, optionally CaCb, at a ratio from about 1 :0.08 to about 1:1 or about 1 :0.1 to about 1 :1. In one embodiment, the ratio may be from about 1 :0.05 to about 1 :1, from about 1 :0.15 to about 1 :1, from about 1 :0.20 to about 1 :1, from about 1 :0.25 to about 1 :1, from about 1 :0.30 to about 1 :1, from about 1 :0.35 to about 1 :1, from about 1 :0.40 to about 1 :1, from about 1 :0.45 to about 1 :1, from about 1 :0.50 to about 1 :1, from about 1 :0.55 to about 1 :1, from about 1 :0.60 to about 1 :1, from about 1 :0.65 to about 1 :1, from about 1 :0.70 to about 1 :1, from about 1 :0.75 to about 1 :1, from about 1:0.80 to about 1:1, from about 1 :0.85 to about 1:1, from about 1:0.90 to about 1:1, or from about 1:0.95 to about 1:1. In one embodiment, the first component comprises the bacterium and soluble Ca²⁺ salt, optionally CaCb, at a ratio of about 1:0.05, about 1:0.1, about 1:0.15, about 1:0.2, about 1:0.25, about 1:0.3, about 1:0.35, about 1:0.4, about 1:0.45, about 1:0.5, about 1:0.55, about 1:0.6, about 1:0.65, about 1:0.7, about 1:0.75, about 1:0.8, about 1:0.85, about 1:0.9, or about 1:0.95.

In one embodiment, the second component comprises urea, a soluble Ca²⁺ salt, and yeast extract at a ratio of about 1 :1.5:0.30 to about 1 :1.8:0.35. In one embodiment, the ratio of urea, a soluble Ca²⁺ salt, and yeast extract may be 1:x:y, where x is selected from about 1.50, about 1.51, about 1.52, about 1.53, about 1.54, about 1.55, about 1.56, about 1.57, about 1.58, about 1.59, about 1.60, about 1.61, about 1.62, about 1.63, about 1.64, about 1.65, about 1.66, about 1.67, about 1.68, about 1.69, about 1.70, about 1.71, about 1.72, about 1.73, about 1.74, about 1.75, about 1.76, about 1.77, about 1.78, about 1.79, or about 1.80, and y is selected independently from about 0.31, about 0.32, about 0.33, about 0.34 or about 0.35. In one embodiment, the ratio may be about 1:1.833:0.333. For example, the ratio may be about 1:1.8:0.3, about 1:1.8:0.31, about 1:1.8:0.32, about 1:1.8:0.33, about 1:1.8:0.34, about 1:1.8:0.35, about 1:1.50:0.3, about 1:1.51:0.3, about 1:1.52:0.3, about 1:1.53:0.3, about 1:1.54:0.3, about 1:1.55:0.3, about 1:1.56:0.3, about 1:1.57:0.3, about 1:1.58:0.3, about

1:1.59:0.3, about 1:1.60:0.3, about 1:1.61:0.3, about 1:1.62:0.3, about 1:1.63:0.3, about

1:1.64:0.3, about 1:1.65:0.3, about 1:1.66:0.3, about 1:1.67:0.3, about 1:1.68:0.3, about

1:1.69:0.3, about 1:1.70:0.3, about 1:1.71:0.3, about 1:1.72:0.3, about 1:1.73:0.3, about

1:1.74:0.3, about 1:1.75:0.3, about 1:1.76:0.3, about 1:1.77:0.3, about 1:1.78:0.3, about

1:1.79:0.3, about 1:1.80:0.3.

An embodiment of the first aspect provides the first component applied admixed with water. One embodiment further provides a') drying the first component applied to the location of the granular substrate.

An embodiment of the first aspect provides the first component applied dry and the method further comprises a') admixing the first component with water. One embodiment further provides a") drying the first component applied to the location of the granular substrate.

In one embodiment, the second component is applied admixed with water.

In another embodiment, the second component is applied dry and the method further comprises b') admixing the second component with water. In one embodiment, the alka lophilic ureolytic calcifying bacterium is of genus Bacillus.

In one embodiment, the alka lophilic ureolytic calcifying bacterium is of species Bacillus clausd' .

In one embodiment, the alkalophilic ureolytic calcifying bacterium is halo-alkalophilic. In one embodiment, the alkalophilic ureolytic calcifying bacterium is halo-alkalophilic and the medium has about pH 10 and further comprises about 2% to about 10% NaCI.

In one embodiment, the alkalophilic ureolytic calcifying bacterium is Bacillus clausd' S111 deposited as accession number V20/026707.

In one embodiment, the halo-alkalophilic ureolytic calcifying bacterium is Bacillus clausd' T09054 deposited as accession number V20/026706.

The bacterium Bacillus clausd' T09054 was deposited at the National Measurement Institute, 1/153 Bertie Street, Port Melbourne, Victoria, 3207, Australia on 7 December 2020 with the accession number V20/026706.

The bacterium Bacillus clausd' S111 was deposited at the National Measurement Institute, 1/153 Bertie Street, Port Melbourne, Victoria, 3207, Australia on 7 December 2020 with the accession number V20/026707.

In one embodiment, the method comprises drying the first component applied to the location of the granular substrate for at least 2 hours.

In one embodiment, the method comprises drying the second component applied to the location of the granular substrate. In one embodiment, drying the second component applied to the location of the granular substrate comprises drying for at least 12 hours.

One embodiment comprises repeating a) and b).

One embodiment comprises a) applying the first component at from about 0.10 L/m² to about 0.15 L/m². In one embodiment the first component may be applied at about 0.5 L/m², about 0.6 L/m², about 0.7 L/m², about 0.8 L/m², about 0.9 L/m², about 0.1 L/m², about 0.11 L/m², about 0.12 L/m², about 0.13 L/m², about 0.14 L/m², about 0.15 L/m², about 0.16 L/m², about 0.17 L/m², about 0.18 L/m², about 0.19 L/m², or about 0.2 L/m².

In one embodiment, applying comprises pouring and/or spraying.

In one embodiment, the first component and/or second component further comprises perlite.

One embodiment further comprises combining the first component with perlite, and/or coating perlite with the second component. In one embodiment, the first component is combined with cement at a ratio from about 0.7:3.0 to about 1.4:3.0 in aqueous solution or suspension. In one embodiment the first component is combined with cement at a ratio from about 0.8:3.0 to about 1.4:3.0, from about 0.9:3.0 to about 1.4:3.0, from about 1.0:3.0 to about 1.4:3.0, from about 1.1 :3.0 to about 1.4:3.0, from about 12:3.0 to about 1.4:3.0, or from about 1.3:3.0 to about 1.4:3.0. The first component may be combined with cement at a ratio of about 0.5:3.0, about 0.6:3.0, about 0.7:3.0, about 0.8:3.0, about 0.9:3.0, about 1.0:3.0, about 1.1 :3.0, about 12:3.0, about 1.3:3.0, about 1.4:3.0, about 1.5:3.0, or about 1.6:3.0.

In one embodiment, the cement comprises a cement mortar additive and the cement mortar additive is present in the cement at from about 100 g/kg to about 200 g/kg of cement in perlite additive form. In one embodiment, the cement mortar additive is present in the cement at about 90 g/kg, about 100 g/kg, about 110 g/kg, about 120 g/kg, about 130 g/kg, about 140 g/kg, about 150 g/kg, about 160 g/kg, about 170 g/kg, about 180 g/kg, about 190 g/kg, about 200 g/kg, or about 210 g/kg.

A second aspect provides a kit when used according to the method of the first aspect, the kit comprising a first component comprising alkalophilic ureolytic calcifying bacterium and a soluble Ca²⁺ salt, optionally CaCb, and a second component comprising urea, a soluble Ca²⁺ salt, optionally CaCb, and yeast extract.

A third aspect provides a kit comprising a first component comprising an alkalophilic or ha lo-alka lophilic ureolytic calcifying bacterium and a soluble Ca²⁺ salt, optionally CaCb, and a second component comprising urea, a soluble Ca²⁺ salt, optionally CaCb, and yeast extract, wherein the alkalophilic ureolytic calcifying bacterium is Bacillus clausd' S111 deposited as accession number V20/026707 or Bacillus clausd' T09054 deposited as accession number V20/026706.

In one embodiment, the kit comprises instructions for use. In one embodiment, the kit comprises instructions for use according to the method of the first aspect.

In one embodiment of the second or third aspects, the first component and/or the second component is admixed with water.

In one embodiment of the second or third aspects, the first component and/or the second component is dry for admixture with water.

A fourth aspect provides a biocement when formed according to the method of the first aspect. A fifth aspect provides a biocement comprising alka lophilic ureolytic calcifying bacterium Bacillus clausd' S111 deposited as accession number V20/026707 or Bacillus clausd' T09054 deposited as accession number V20/026706.

In any embodiment of the aspects above, yeast extract may be substituted for a nutrient broth.

Definitions

The term "granular substrate" as used herein refers to a substance of macroscopic particles. As used herein, granular substrate may refer to a cementitious substrate, e.g., mortar, wall, soil, sand, clay, flyash, or a coating of cement on another substrate, for example.

As used herein, "location of a granular substrate" indicates that the first and second component need not be applied to the entire granular substrate, but may be applied only to a sub-portion of the granular substrate. Generally, although not exclusive, this would be a location in need of remediation. Nevertheless, the term "location of a granular substrate" does not exclude the embodiment in which the first and second component are applied to the entire granular substrate.

As used herein, the term "applying" and variations thereof is used inclusively, for example the term contemplates adding, spraying, and mixing.

The term "biocementation" as used herein refers to the biological process of carbonate production and precipitation by an organism in a cementitious environment.

As used herein, "cementitious" refers to a material that has the nature of cement.

The term "compressive strength" as used herein refers to the ability of a substance to resist breaking under compression.

The term "a Ikalophilic" as used herein means an organism capable of optimally surviving in an extreme alkaline environment, i.e., where the pH is greater than 8.5.

The term "ha lo-alka lophilic" as used herein means an organism capable of optimally surviving in an extreme alkaline and saline environment, i.e., where the pH is greater than 9 and the salt concentration is greater than 2% w/v. The term "extremophilic environment" as used herein means an environment suitable for the optimal growth of an organism that would be considered extreme for carbon-based life form. As used herein, extremophilic environments include high pH and high pH and high salinity.

The term "ureolytic" as used herein means an organism that is capable of enzymatically breaking down urea to produce ammonia and carbonate.

The term "calcifying" as used herein means an organism that is capable of producing and excreting an insoluble calcium salt.

The term "lyophilised" as used herein refers to the process of dehydration performed via a process that includes freezing a substance, lowering pressure and removing ice by sublimation, i.e., transition directly from solid to gas.

The term "perlite" as used herein refers to an amorphous volcanic glass that has a relatively high-water content. Perlite may be a commercial grade perlite. Perlite may be a product with the CAS number 93763-70-3.

The term "coating" as used herein means the application of a substance onto the surface of a product. As used herein, the coating of a particle was via the spraying of a first solution onto said particle.

The term "kit" as used herein means refers the combination of at least one component used for the production of a biocementation product. Optionally the kit may include instructions outlined how to use said kit.

The term "self-healing" as used herein means refers to the ability of a granular substrate comprising a mixture of a combination of an admixture of the first and second component and a binder agent. Upon contact with water, the mixture comprising the first component, second component and binder agent can increase the compressive strength of the surrounding area by precipitating CaCOs cement.

The term "about" as used herein means that the parameter may vary by as much as 10% below or above the stated numerical value for that parament. For example, the pH of about 10.0 may vary between pH 9.0 and pH 11.0

In the claims which follow and in the description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

Examples 1. Enrichment

Alka lophilic or halo-alkalophilic ureolytic calcifying bacterial cultures were enriched and isolated from calcareous soil, cement and thrombolites collected in Western Australia. The enrichments were done under extreme cementitious environments of pH 8.5-13 or pH 10 and salinity (NaCI) ranges from 1-10% w/v. The enrichment media is shown in table 1 below. Table 1: Constituents of media used (per litre)

Briefly, 1 g of soil, cement, and thrombolites were inoculated in 100 ml of the above MSM media in 500 ml flasks and left to grow at 37 °C and 100 rpm for 2 weeks. Subsequent sub culturing with 1 % inoculum were done in fresh media every 2-3 weeks. At least three sub culturing were done.

A second media used for enrichments of a Ikalophilic ureolytic bacteria was Cement extract solution media. For this, 100 g cement was mixed in 1 litre of deionized water and stirred continuously at 30 rpm on magnetic shaker for 1 hour. The cement suspension was filter sterilised and then supplemented with 2 % yeast extract (20 g/l) and 5 % urea (48 g/l) at pH 12 to make alkalophilic Cement extract media (A-CEM) or pH10 and 2 % salinity (2 g NaCI/100 ml) to make halo-alkalophilic CEM (H-CEM). Again, 1 g of soil, cement, and thrombolites samples were added in the A-CEM or H-CEM separately for enrichment and isolation of alkalophilic or halo-alkalophilic bacteria as done previously in MSM media.

2. Isolation

In the media sets which showed significant bacterial culture growths after 3-4 enrichments compared to the controls with clear media, the cultures were plated on alkalophilic media plates (AIM) at pH 10, 11, 12 and 13 for alkalophiles and on haloalkaliphilic media plates (HIM) at pH 10 with 2.5%, 5%, 7.5% NaCI for halo-alkalophiles (see table 2) and sub cultured thrice.

Table 2: Constituents of Alkalophilic isolation media (AIM) and haloalkalophilic isolation media (HIM) used (per litre)

Bacterial colonies which grew on high pH or high pH and salt plates were then picked up and tested for their qualitative ureolytic activities on urea agar base plates to check the production of urease based on the intensity of pink color, which can be checked easily by naked eyes. Based on this qualitative method, a particular colony showing the best pink colour intensity on the medium was isolated, cultured on AIM or HIM overnight for isolation of its DNA. The culture was then characterised for its genotypic identification. 16S rRNA gene sequencing was conducted to identify the cultures and their details were submitted in NCBI database.

The cultures were then checked for growth, viability, urease activity, urease stability, physiological characteristics, calcium carbonate precipitation ability. All the experiments were performed in triplicates.

3. Growth of cultures under cementitious environment

The following example demonstrates high growth, viability, urease activity of the isolated a Ika lophi lie and ha loa Ika lophilic ureolytic cultures Bacillus clausd' S111 and Bacillus clausd' T09054 compared to the standard Sporosarcina pasteurd' ATCC 11859.

The culture Bacillus clausd' S111 was grown in AIM at pH 11 and culture of Bacillus clausii T09054 was grown in HIM at pH 9 (2.5 % NaCI) along with S. pasteurd' ATCC11859 in its specific YE-AS media in 100 ml media within 500 ml conical flasks. Briefly, all the cultures were grown overnight in specific media conditions at 37 °C and 180 rpm to reach an optical density of 1 (OD600 nm). 1% of the inoculum from each flask was further sub cultured into 100 ml of fresh media of AIM or HIM. The growth curves of the cultures were studied by measuring the optical density (OD600 nm) using a spectrophotometer and results are demonstrated in figure 1.

4. Viability under cementitious environment

For estimation of bacterial cell viability, Bacillus clausd' S111, Bacillus clausd' T09054, and S. pasteurd' cultures were grown overnight in their specific growth medias to OD 1 (OD600 nm). They were then transferred to a Ikalophilic Cement extract media (A-CEM) - pH 12 (without yeast extract) and halo-alkalophilic Cement extract media (H-CEM) - pH 9 (with 2 % NaCI and without yeast extract) prepared in 500 ml conical flasks comprising 100 ml of the media at an initial cell concentration of 0.2 OD (OD600 nm) and monitored for their viability in the next 5 days. The cell viability was monitored by ATPase detection assay. The assay is performed using BacTiter-Glo™ from Promega, 100 pl of bacterial sample was mixed 100 pl of reagent and luminescence is measured using a luminometer over a period of time. This assay measures the luminescent signals which are directly proportional to the number of viable cells in the culture. The results are demonstrated in figure 2.

5. Urease activity

Bacillus clausd' S111, Bacillus clausd' T09054, and S. pasteurd' cultures were grown up to OD 0.5 (OD600 nm) in their specific medias. 1 % inoculum was inoculated in 250 ml flasks containing 50 ml of AIM at pH 11 and HIM at pH 9 and 5 % NaCI. After inoculation, the flasks were incubated at 37 °C on a rotary shaker at 130 rpm for different time intervals (24, 48, 72, 96, 120, 144 and 168 hrs.). The above culture broths were centrifuged at 8000 rpm for 5 minutes at 4 °C to collect the supernatant and check the extracellular urease activity. 2.5 ml supernatant was incubated with 1 ml substrate urea (0.1 M) and 1 ml potassium phosphate buffer (pH 8) at 37 °C for 5 minutes followed by an additional incubation at 37 °C for 5 minutes in 1 ml phenol nitroprusside and 1 ml alkaline hypochlorite. One unit of urease activity is defined as the amount of enzyme hydrolysing one pmole urea per min. Triplicates were maintained for these experiments. The results are demonstrated in figure 3.

6. Calcium carbonate precipitation profile

The cementation medium used in this study contains 2 g/l of yeast extract, 30 g/l urea, and 55 g/l CaCl2-2H2O. 65 mL of deionized water containing 2 g of yeast extract was prepared and pH adjusted to 8.0 with 1 M NaOH solution and autoclaved separately. Then 5 M and 2 M filter-sterilized urea and CaCl2-2H2O stock solutions were prepared, respectively. From the stock solution, 10 ml of urea, and 25 ml CaCl2-2H2O were added into the autoclaved yeast extract solution to achieve a final concentration of 0.5 M urea and CaCl2-2H2O. The overnight culture of Bacillus clausd' S111 grown in AIM at pH 11, Bacillus clausii T0905 grown in HIM at pH 9 and 2.5 % NaCI along with S. pasteurd' ATCC11859 grown in AIM at pH 11 and HIM was centrifuged at 8000 rpm for 5 mins at 4 °C, and the pellet was inoculated into the cementation medium and incubated at 37 °C and 180 rpm for 5 days. The samples (5 ml) were taken from the cementation medium over a period of 5 days, centrifuged at 3000 g for 10 minutes at 4 °C and the supernatant was used to measure concentrations of soluble calcium ions. At the end of 5 days, the pellet retrieved from the culture after centrifugation was washed twice with distilled water, dried at 70 °C for overnight and used to measure the dry weight of the CaCOs.

The complexometric titration method was used to estimate the soluble concentration of Ca²⁺ in the supernatant. 40 pl of supernatant was diluted into 10 ml and 400 pl of

1 M NaOH was added to the solution to raise the pH to around 10. After that, a few drops of hydroxy naphthol blue disodium salt (1 % W/V) solution was added as an indicator. The titration was performed against 1 mM EDTA disodium salt solution until the colour changes from pink to blue, and the endpoints were noted. In this study, the standard solutions of 0 to

2 mM CaCb were prepared and titrated against 1 mM titrant solution. The endpoints of the standard solutions were noted. A graph was plotted between the concentrations of CaCb solution vs. the volume of 1 mM EDTA required to reach the endpoint. An unknown sample concentration was found from the slope of the plot. The results for soluble and insoluble Calcium ions and CaCOs precipitates are demonstrated in figure 4 and 5.

7. Physiological characteristics cultures of Bacillus clausd' S111 and Bacillus clausd' T0905 were tested for their physiological characteristics.

The results are demonstrated in table 3.

8. Bioconcrete Sealant with improved bacterial fixation

Bioconcrete Sealant is a two-component low viscosity, eco-friendly, biocement solution manufactured using soil bacteria for sealing of cracks up to 2.0 mm and grouting in deep pours up to 500 mm. a. As lyophilised powder for concrete cracks

Alkalophilic or halo-alkalophilic ureolytic calcifying bacterial culture is centrifuged at 3000 g for 10 mins at 4 °C and the pellet re-suspended in sterile water at a final concentration of 10⁹-10¹⁰ cells per ml. 100 ml of suspension was transferred into a container pre-cooled in liquid nitrogen and kept inside the freeze dryer. After 15 min, the suspension is freeze-dried. The temperature of the freeze-dryer shelf was maintained at -50 °C for 4 h; increased to -5 °C over 15 h; maintained at -5 °C for 6 to 12 h; and increased to -3 °C over 4 h. The lyophilised cell powder was homogenised. 1 g of the powder was mixed with 0.08 - 1.0 g CaCb and called as component A. The powder was stored in a cool place.

120 g Urea and 220 g CaCb were mixed and the mixed powder, called component B, was stored in a cool place.

5.0 - 5.5 g of component A is mixed in 1 litre of water and stirred continuously at room temperature with a stirring rod and separately, 85 g of component B is mixed into 1 litre water by stirring continuously, admixed component A is applied into concrete cracks by pouring or spraying for general surface treatment. The cracked concrete is left to dry for 2-3 hours. Admixed component B is applied to the dried concrete. The concrete is left to dry for 12-16 hours. If required, add admixed component B at intervals of 12-16 hours. Depending upon the size of the cracks/surface treatment intensity, multiple applications of admixed component B are provided to complete plugging. The concrete specimens were left to dry in oven for 24 hours at 40 °C before further testing.

Typical application of admixed component A is 0.10 - 0.15 L/m² for general surface repair and 0.5 - 0.6 L/m² for crack repair. Specific consumption is determined by measuring the test area. Multiple treatments might be required for wider cracks and can be applied with an interval of one week.

Bioconcrete Sealant was 90 % more effective than conventional grouts, cement, chemical polymers in sealing cracks up to 2 mm. b. In carrier for self-healing concrete

Perlite powders were dried in the oven at 40 °C for 1 day until a constant weight was obtained. Dried bacterial cell pellets (cells and spores) of ~ 10¹⁰ cells/ml and 0.08 - 0.1 g CaCb were mixed with 5 g of perlite powder. The mixture was further dried at 45 °C to attain a constant weight. After this, 10 ml of a solution containing urea 6 g/l, 11 g/l CaCb, 2 g/l Yeast extract was sprayed on the surface of the particles and further subjected to 45 °C for 2 days in the oven.

Bioperlite additive is mixed into the cement at a ratio of 0.7 - 1.4:3.0 in aqueous solution or suspension form. Preferably, the quantity of the said cement mortar additive in the cement is 100 - 200 g/kg of cement in perlite additive form. After mixing the said additive, the mortar should be used and cured as per the standard practice or as recommended by the manufacture of the cement.

Bioperlite cement mortar has 25-30% more compressive strength and 65-75% more self - healing ability for sealing of cracks than untreated mortar.

9. Self-healing admixture

This is a general-purpose admixture that is a precursor to other products such as self- healing sealant and self-healing repair mortar. This can be used in concrete, mortar and soil for achieving self-healing ability. The admixture is a powder that can be mixed with binders such as cement, fly ash, and other supplementary cementitious materials before mixing and casting. After solidification, in case of any cracking of a parent material, the admixture activates in contact of water and heals the damage. It also densifies the surrounding area and prevents further damage.

The admixture contains lyophilized bacteria and other chemicals encapsulated in perlite particles of 0.25 to 0.5 mm diameter. a. As encapsulated powder

Perlite powders were dried in the oven at 40 °C for 1 day until a constant weight was obtained. Dried bacterial cell pellets (cells and spores) of ~ 10¹⁰ cells/ml and 0.08 - 0.1 g CaCb was mixed with 5 g of perlite powder. They were further dried at 45 °C to attain a constant weight. After this, a 5ml solution containing urea 6g/l, 11 g/l CaCb, 2g/l Yeast extract was sprayed on the surface of the particles and further subjected to 45 °C for 2 days in the oven. The self-healing powder is dry mixed with the binder at a ratio of 0.1 - 1 % by weight. The mixture is turned thoroughly with a trowel for about 3 minutes to ensure uniform dispersion. The mixture can be added to the concrete or mortar mix as ordinary cement.

The self-healing admixture heals cracks of width 0.01 - 1 mm. The healed concrete regains the same level of resistance to permeation of moisture as the undamaged concrete.

10. Bioconcrete Repair Mortar: Self-healing self-sensing

Bioconcrete Repair Mortar is a cement based ready to mix repair mortar for self- healing structural repairs in concrete. a. As paste for concrete surface

Lyophilised calcifying bacterial culture cells were produced as described above. Self- healing admixture (containing 1 - 1.2 g cell powder, 0.08 - 0.1 g CaCb, urea 6 g/l, 11 g/l CaCb, and 2 g/l Yeast extract, as described above in 9) is mixed with silica sand, cement, silica fume, self-sensing admixture (carbon fibre), ground granulated blast-furnace slag (GGBFS) in the following ratio:

For repair mortar applications, this mixture is added to water with Water/binder ratio of 0.3 - 0.4:1 and shaken for mixing. This self-healing, self-sensing repair mortar paste is then applied to a granular surface of concrete.

Bioconcrete Repair mortar has 25 - 30 % more compressive strength and 70 - 80 % more self-healing ability for sealing of cracks than the untreated mortar.

Claims

Claims The claims defining the invention are as follows:

1. A method of biocementation, comprising: a. applying to a location of a granular substrate a first component comprising an alkalophilic ureolytic calcifying bacterium and a soluble Ca²⁺ salt in a medium having about pH 10 to about pH 13; and b. applying to the location of the granular substrate a second component comprising urea, a soluble Ca²⁺ salt, and yeast extract, wherein the first component is applied to the location of the granular substrate separately to the second component.

2. The method of claim 1, wherein the soluble Ca²⁺ salt is CaCb, Ca NC h, CaSC , and Ca(C2HsO2)2, or a combination thereof.

3. The method of claim 1 or 2, wherein the bacterium is lyophilised, and wherein the first component comprising bacterium and a soluble Ca²⁺ salt is mixed at a ratio from about 1 :0.08 to about 1 :1, respectively, and wherein the second component comprising urea, a soluble Ca²⁺ salt, and yeast extract is mixed at a ratio from about 1 :1.8:0.3, respectively.

4. The method of any one of claims 1 to 3, wherein the first component is applied admixed with water and the method further comprises a') drying the first component applied to the location of the granular substrate.

5. The method of any one of claims 1 to 3, wherein the first component is applied dry and the method further comprises a') admixing the first component with water and a") drying the first component applied to the location of the granular substrate.

6. The method of any one of claim 1 to 5, wherein the second component is applied admixed with water. The method of any one of claim 1 to 5, wherein the second component is applied dry and the method further comprises b') admixing the second component with water. The method of any one of claims 1 to 7, wherein the alka lophilic ureolytic calcifying bacterium is of genus Bacillus. The method of claim 8, wherein the a I ka lophi lie ureolytic calcifying bacterium is of species Bacillus clausd' . The method of any one of claims 1 to 9, wherein the alkalophilic ureolytic calcifying bacterium is halo-alkalophilic. The method of claim 10, wherein the halo-alkalophilic ureolytic calcifying bacterium and the medium has about pH 10 and further comprises about 2% to about 10% NaCI. The method of any one of claims 1 to 9, wherein the alkalophilic ureolytic calcifying bacterium is Bacillus clausd' S111 deposited as accession number V20/026707. The method of claim 10 or 11, wherein the halo-alkalophilic ureolytic calcifying bacterium is Bacillus clausd' T09054 deposited as accession number V20/026706. The method of claim 3 or 4, comprising drying for at least 2 hours. The method of claim 5 or 6, further comprising drying the second component applied to the location of the granular substrate. The method of claim 15, comprising drying for at least 12 hours. The method of any one of claims 1 to 16, comprising repeating a) and b). The method of any one of claims 1 to 17, comprising a) applying the first component at from about 0.10 L/m² to about 0.15 L/m². The method of any one of claims 1 to 18, wherein applying comprises pouring and/or spraying. The method of any one of claims 1 to 19, wherein the first component and/or second component further comprises perlite. The method of any one of claims 1 to 20, further comprising combining the first component with perlite, and/or coating perlite with the second component. The method of any one of claims 1 to 21, wherein the first component is combined with cement at a ratio from about 0.7:3.0 to about 1.4:3.0 in aqueous solution or suspension. The method of claim 22, wherein the cement comprises a cement mortar additive and the cement mortar additive is present in the cement at from about 100 g/kg to about 200 g/kg of cement in perlite additive form. A kit when used according to the method of any one of claims 1 to 23, the kit comprising a first component comprising alkalophilic ureolytic calcifying bacterium and a soluble Ca²⁺ salt and a second component comprising urea, a soluble Ca²⁺ salt, and yeast extract. A kit comprising a first component comprising an alkalophilic ureolytic calcifying bacterium and a soluble Ca²⁺ salt, and a second component comprising urea, a soluble Ca²⁺ salt, and yeast extract, wherein the alkalophilic ureolytic calcifying bacterium is Bacillus clausd' S111 deposited as accession number V20/026707 or Bacillus clausd' T09054 deposited as accession number V20/026706. The kit of claim 24 or 25, wherein the first component and/or the second component is admixed with water. The kit of claim 24 or 25, wherein the first component and/or the second component is dry for admixture with water. A biocement when formed according to the method of any one of claims 1 to 23. A biocement comprising a Ika lophi lie ureolytic calcifying bacterium Bacillus clausd' S111 deposited as accession number V20/026707 or Bacillus clausd' T09054 deposited as accession number V20/026706. Bacillus clausd' T09054 deposited as accession number V20/026706. Bacillus clausd' S111 deposited as accession number V20/026707.

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